Antifade solution

Shrimp hemocytes were collected and separated on poly-l-lysine-coated glass slides (Sigma), followed by standing for 10 min at 4°C. The hemocytes were fixed in 4% paraformaldehyde for 25 min at 4°C. The fixed hemocytes were washed with cold PBS and then subjected to the permeabilization with 0.2% Triton X-100 for 5 min. Next, the hemocytes were equilibrated in 100 µl of equilibration buffer at 4°C for 10 min. The equilibrated hemocytes were counterstained with PI after incubation with rTdT mix in a humid environment for 1 h. Subsequently, 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) was added to the slide to stop the reaction. The slide was covered with antifade solution (Invitrogen) to prevent signal quenching.

TUNEL system (Promega) was conducted according to the manufacturer's instruction with some minor modifications. Briefly, the medium of cells was removed after the indicated treatment. The cells were fixed with 1% methanol‐free paraformaldehyde for 10 min at 4 °C. Subsequently, the cells were rinsed twice with PBS and permeabilized with 0.1% Triton X‐100 for 5 min. After rinsing slides with PBS twice at room temperature, the cells were covered with 100 μL of equilibration buffer at room temperature for 10 min. The cells were incubated with rTdT mix containing green fluorescein‐12‐dUTP for 60 min at 37 °C and then counterstained with PI. The reactions were terminated by immersing the slide in 2× SSC for 15 min. The slide was air‐dried and mounted with antifade solution (Invitrogen) to assess fluorescence by microscopy.

Cells grown on coverslips were fixed with freshly made 3% paraformaldehyde/2% sucrose solution for 10 min after CPT treatment. For chromatin-bound MUS81, RAP32-pS4/8, and S9.6 staining, cells were pre-extracted before fixation using pre-extraction buffer (10 mM PIPES pH 6.8, 100 mM sodium chloride, 300 mM sucrose, 3 mM magnesium chloride, 1 mM EGTA, 0.2% Triton X-100). After fixation, cells were permeabilized with 0.5% Triton X-100 solution for 5 min on ice, washed with phosphate-buffered saline (PBS), and stained with indicated primary antibodies diluted in 1% bovine serum albumin (BSA) at 37 °C for 1 h followed by incubation with secondary antibodies conjugated with Alexa-488 (Invitrogen, A11008, 1 : 1000 dilution) or Alexa-555 (Invitrogen, A31570, 1 : 1000 dilution) for 1 h at 37 °C. Coverslips were then washed with PBS and mounted using 4′,6-diamidino-2-phenylindole (DAPI) containing antifade solution (Invitrogen). For cells pulse-labeled with EdU (10 µM) and IF staining, cells were fixed, permeabilized, and stained with indicated primary and secondary antibodies, followed by EdU-click reaction using Click-iT EdU Alexa Fluor 488 imaging kit. Images were collected with 80i eclipse Nikon microscope using ×40 or ×63 objective using NIH Elements AR software (AR 5.10.01 64 bit software).